Recombinant Mouse Lipoma-preferred partner homolog (Lpp)

What is Mouse Lipoma-preferred partner homolog (Lpp) and what are its key genetic identifiers?

Mouse Lipoma-preferred partner homolog (Lpp) is a LIM domain-containing protein that functions as a focal adhesion molecule involved in cell adhesion and migration dynamics. The gene has multiple identifiers including C79715, AA959454, AU024130, D630048H16, 9430020K16Rik, and B130055L10Rik . It is also referred to as "lipoma-preferred partner homolog isoform 1" or "LIM domain containing preferred translocation partner in lipoma" in scientific literature .

The protein's significance stems from its involvement in cellular adhesion complexes and its interaction with key regulatory proteins such as protein phosphatase 2A (PP2A) . From a structural perspective, mouse Lpp contains three LIM domains that serve as critical protein-protein interaction modules, particularly important for its binding with other cellular components.

How do the LIM domains of Mouse Lpp contribute to its functional properties?

The Lpp protein contains three LIM domains (located at residues 415-612) that play distinct but complementary roles in protein-protein interactions . Research demonstrates that these domains have differential contributions to binding:

Experimental evidence indicates that while all three LIM domains contribute to interaction with binding partners such as PR130, the integrity of LIM2 and LIM3 domains appears more critical for these interactions in the context of the full-length protein . When researchers mutated four structurally important cysteine or histidine residues to alanine in each LIM domain individually, mutations in LIM2 or LIM3 abolished binding to PR130, while the LIM1 mutant retained partial binding capacity .

What expression systems are optimal for producing functional Recombinant Mouse Lpp?

Multiple expression systems can be employed for producing Recombinant Mouse Lpp, each with distinct advantages depending on your experimental requirements:

According to available product information, Recombinant Mouse Lpp can be successfully produced in all four expression systems with purity typically reaching ≥85% as determined by SDS-PAGE analysis . The choice of expression system should align with your experimental objectives, particularly considering whether post-translational modifications are critical for your functional studies.

What quality control methods should be employed to verify recombinant Lpp integrity?

For rigorous quality assessment of Recombinant Mouse Lpp, researchers should employ multiple complementary techniques:

• SDS-PAGE analysis: Standard for assessing purity, with recommended threshold of ≥85% purity . This should be accompanied by western blotting using specific anti-Lpp antibodies.

• Functional binding assays: Given Lpp's known interaction with PP2A regulatory subunits, co-immunoprecipitation assays with PR130/B′′α1 can verify functionality .

• LIM domain integrity assessment: Since the LIM domains are critical for interactions, circular dichroism (CD) spectroscopy to verify proper folding, particularly of zinc finger motifs within these domains.

• Endotoxin testing: Similar to other recombinant proteins (e.g., the TNF standard with endotoxin levels ≤0.1 ng per μg protein ), Lpp preparations should undergo LAL chromogenic assay to ensure minimal endotoxin contamination for cell-based experiments.

How should researchers design experiments to study Lpp's role in cell adhesion and migration?

When investigating Lpp's functions in cellular adhesion and migration, consider these methodological approaches:

Cell Adhesion Dynamics Analysis:

• Focal adhesion visualization: Transfect cells with fluorescently-tagged Lpp constructs (wild-type and domain mutants) and employ live-cell imaging to monitor focal adhesion formation dynamics.

• Binding partner depletion: Design experiments using PR130 knockdown or knockout models, as research demonstrates that complex formation between LPP and PR130-PP2A is mandatory for cell adhesion functions .

• Domain-specific functional rescue: When working with Lpp-depleted cells, compare rescue experiments using wild-type Lpp versus LIM-domain mutants, particularly focusing on LIM2 and LIM3 domains that are critical for binding to PR130 .

Migration Assays:
Implement multiple complementary migration assays (wound healing, transwell, and single-cell tracking) to comprehensively assess how Lpp influences different aspects of cell migration. Current literature indicates that PP2A binding through PR130 is essential for Lpp's functions in migration dynamics , suggesting experiments should include conditions that modulate this interaction.

What are the critical considerations when designing experiments to study Lpp-PP2A interactions?

Research has established that Lpp specifically interacts with PP2A through the PR130/B′′α1 regulatory subunit . When designing experiments to investigate this interaction:

• Construct design: Create a panel of Lpp constructs including:

  • Full-length protein

  • Isolated LIM domains (residues 415-612)

  • Non-LIM region (residues 1-415)

  • Individual LIM domain mutants (particularly focusing on structurally important cysteine/histidine residues)

• Interaction analysis:

  • Co-immunoprecipitation with PR130 should be performed with each construct

  • Yeast two-hybrid validation can confirm direct interactions

  • Quantitative binding assays (e.g., surface plasmon resonance) can determine binding kinetics

• Functional readouts:

  • Phosphatase activity assays to assess how Lpp binding affects PP2A catalytic function

  • Cell adhesion and migration assays comparing wild-type versus Lpp binding-deficient PR130 mutants

A critical experimental control, as demonstrated in the literature, is the use of PR130 mutants that no longer bind to Lpp, as these fail to rescue phenotypes in PR130-depleted cells .

How can Lpp be employed in cancer research models?

Lpp was originally identified in the context of lipomas (hence its name "lipoma-preferred partner"), and its role in cell adhesion and migration suggests potential involvement in cancer progression . Research approaches should:

• Expression profiling: Analyze Lpp expression across cancer types and correlate with clinical outcomes, particularly in cancers where cell migration is a key determinant of progression.

• Functional studies: Implement gain-of-function and loss-of-function approaches in syngeneic mouse models similar to those used for immunology studies (e.g., CT26, MC38, and B16F10 models mentioned in cancer research contexts) .

• Drug response modulation: Investigate whether Lpp expression or activity influences cancer cell response to therapeutics, particularly those targeting adhesion or migration pathways.

• Signaling pathway integration: Map how Lpp-PP2A interactions modulate specific signaling cascades relevant to cancer progression, using phosphoproteomic approaches to identify key substrates.

What methodological approaches should be used to study post-translational modifications of Lpp?

Being a crucial protein in cellular adhesion complexes, Lpp likely undergoes regulatory post-translational modifications. Researchers should:

• Phosphorylation analysis: Given Lpp's interaction with PP2A (a phosphatase) , phosphorylation is likely a key regulatory mechanism:

  • Use phospho-specific antibodies if available

  • Employ mass spectrometry to map phosphorylation sites

  • Create phosphomimetic and phospho-dead mutants to assess functional impact

• Other modifications:

  • LIM domains contain zinc-finger motifs that could be subject to redox regulation

  • Analyze potential ubiquitination sites that might regulate protein turnover

• Context-dependent modifications:

  • Compare modification patterns between normal adhesion conditions and cellular stress

  • Assess how modifications change during dynamic processes like cell migration

What are the optimal storage conditions for maintaining Recombinant Mouse Lpp activity?

Drawing from established protocols for similar recombinant proteins, the following storage recommendations should be applied to Recombinant Mouse Lpp:

• Upon initial thawing: Aliquot into polypropylene microtubes and freeze at -80°C for future use .

• For extended storage:

  • Dilute in sterile neutral buffer containing carrier protein (0.5-10 mg/mL human or bovine serum albumin)

  • For biological assays: use carrier protein concentrations ≥1 mg/mL

  • For use as standards in immunoassays: use carrier protein concentrations of 5-10 mg/mL

  • Avoid dilution below 50 μg/mL

• Working with purified protein: Handle on ice and minimize freeze-thaw cycles, as LIM domain proteins containing zinc finger motifs can be sensitive to structural disruption.

• Quality control: Periodically verify protein integrity using SDS-PAGE and functional binding assays, especially after extended storage periods.

What troubleshooting strategies should be employed when Lpp binding assays fail?

When encountering difficulties with Recombinant Mouse Lpp binding assays:

• Protein integrity verification:

  • Confirm zinc content using atomic absorption spectroscopy, as zinc is essential for proper LIM domain folding

  • Assess protein aggregation state using dynamic light scattering

  • Verify molecular weight using mass spectrometry to ensure no truncation has occurred

• Binding partner considerations:

  • For PR130 interaction studies, ensure the full three-LIM domain region (residues 415-612) is intact, as deletion of any single LIM domain inhibits binding

  • Check expression levels of binding partners, as overexpression might affect localization

• Experimental conditions optimization:

  • Adjust buffer composition, particularly zinc concentration

  • Optimize protein concentrations to avoid non-specific interactions

  • Consider detergent types and concentrations when working with membrane-associated complexes